| Objective:In previous studies conducted by our research group,protein and gene expression analysis of 14-3-3ζand OPN were performed on tissue samples from cholangiocarcinoma(CCA)patients,revealing that 14-3-3ζ and OPN may together promote invasion and metastasis of CCA.Building upon this work,our current study established an in vitro epithelial mesenchymal transition(EMT)model using CCA cell lines with different levels of 14-3-3ζ expression.Changes in EMT markers(E-cadherin,N-cadherin,β-catenin,and Vimentin)were measured before and after EMT induction,as well as the effects of varying 14-3-3ζ expression on CCA cell migration and invasion.Furthermore,we investigated the specific changes in OPN glycosylation patterns following 14-3-3ζ-mediated EMT in human CCA cells.Subsequently,the GalNAc-T3 glycosyltransferase was screened,its expression in CCA was detected,and the relationship between GalNAc-T3 expression and clinical pathology and prognosis was analyzed,to provide a scientific basis for elucidating the mechanism of early migration and invasion in CCA.Methods:1.Human CCA cell lines(HuCCT1 and TFK-1)were cultured,and 14-3-3ζ-siRNA plasmids were constructed and transfected into them to establish stable cell lines with high or low expression of 14-3-3ζ.The expression of 14-3-3ζ was detected using quantitative real time polymerase chain reaction(qRT-PCR)and Western blot analysis.2.Construct in vitro EMT models of human CCA cell lines with different expressions of 14-3-3ζ,observe the changes in cell morphology under a microscope.Use qRT-PCR and Western blot to detect changes in EMT markers(E-cadherin,N-cadherin,β-catenin,and Vimentin)in 14-3-3ζ+/-human CCA cells before and after EMT.3.The transwell cell migration assay and wound healing assay were used to detect the differences in cell migration and invasion ability with different expressions of 14-3-3ζ.4.Total protein was extracted and OPN was purified using immunoprecipitation techniques.Co-immunoprecipitation(CO-IP)was performed to detect the relationship between 14-3-3ζ and OPN.The expression of 14-3-3ζ and OPN was also detected using qRT-PCR and Western blot analysis in the EMT models of human CCA cells.5.Using lectin microarray technology,detected the specific glycosylation profile of OPN in CCA cells undergoing EMT transformation mediated by 14-3-3ζ.6.Validation of the reliability of lectin microarray results was performed by lectin blot and cellular lectin histochemistry methods.7.The expression of the glycosyltransferase GalNAc-T3 at both the gene and protein levels in samples of CCA and adjacent non-cancerous tissues was investigated using immunohistochemistry(IHC),quantitative real-time polymerase chain reaction(qRT-PCR),and Western blot(WB)analyses in this study.8.The relationship between GalNAc-T3 and the clinicopathological characteristics and prognosis of CCA was investigated by analyzing the clinical and pathological data of patients.Results:1.The expression of 14-3-3ζ was detected in TFK-1 and HuCCT1 human CCA cells transfected with plasmids using qRT-PCR and Western blot analysis.The results showed a significant inhibition of 14-3-3ζ expression in CCA cells.2.Under differential interference contrast microscopy,we observed the cellular morphology of 14-3-3ζ+/-human CCA cells in an in vitro EMT model and found that the cells transformed from columnar epithelial cells to fibroblast-like mesenchymal cells.3.After EMT transformation of 14-3-3ζ+/-human CCA cells,EMT markers(E-cadherin,N-cadherin,β-catenin,and Vimentin)were detected by qRT-PCR and Western blot analysis.It was found that the expression of 14-3-3ζ was significantly inhibited,leading to the suppression of EMT markers.4.The transwell cell migration assay and wound healing assay were conducted to evaluate the changes in CCA cell migration and mobility of 14-3-3ζ+/-human CCA cells undergoing EMT transformation.The results indicated that the migration and mobility of CCA cells were suppressed upon inhibition of 14-3-3ζ expression.5.CO-IP was performed to detect the relationship between 14-3-3ζ and OPN,indicating a binding relationship between 14-3-3ζ and OPN.6.qRT-PCR and Western blot were used to detect the expression of 14-3-3ζ and OPN in the human CCA EMT model.The results showed that both 14-3-3ζ and OPN were highly expressed in the EMT model,suggesting a cooperative role in promoting EMT transformation.7.Lectin microarray was used to analyze the glycosylation profile of OPN in HuCCT1 CCA cells after undergoing EMT.The results showed that the affinity of OPN to lectins LB A,Jacalin,Lotus,PHA-P,ACG,and GAL1 was reduced,while the affinity to lectins SNA-I,GAL1-S,BC2L-A,GAL2,PALa,Con A,and PPL was increased.The glycosylation profile of OPN also showed a decrease in structures such as GalNAc(α1,3)Gaβ,Galβ3Ga1NAc,αFuc,GlcNAcβ4Manα3,α2,3 Sialic Acid,and branched LacNAc,and an increase in structures such as GalNANAα(2,6)Gal,branched LacNAc,High-mannose,GalNAcαl-3Gal,αMan,and α/βGalNAc,suggesting that EMT induction altered the glycosylation pattern of OPN in CCA cells.8.The lectin blotting method was used to validate the lectin chip results.The results showed that the glycan structures bound by LBA lectin were decreased in the high expression 14-3-3ζ biliary cancer cells after EMT transformation.The lectin blotting results were consistent with the chip results,confirming the reliability of the chip results.9.The biotin-labeled lectin LBA chip results were validated using the fluorescence cell lectin immunohistochemistry method.Similar to the chip results,the fluorescence signal intensity of LBA lectin binding to the cell membrane glycoproteins was decreased in the high expression 14-3-3ζ CCA cells after EMT transformation,further confirming the reliability of the chip results.10.GalNAc-T3 was found to be downregulated in CCA based on the expression analysis at the gene and protein levels using immunohistochemistry(IHC),qRT-PCR and WB analyses in CCA and adjacent non-cancerous tissues.11.By analyzing the clinical and pathological data of patients,we found that GalNAc-T3 was expressed at low levels in CCA tissues but at high levels in adjacent non-cancerous tissues.Specifically,GalNAc-T3 expression was low in stage Ⅰ-Ⅱ CCA and in stage Ⅲ-Ⅳ CCA,as well as in well-differentiated and moderately-differentiated tumors.Patients with high expression of GalNAc-T3 had significantly better overall survival than those with low expression.Conclusions:1.The 14-3-3ζ plasmid was successfully used to transfect human CCA cells,establishing an in vitro model of human CCA EMT.In this model,both 14-3-3ζ and OPN were highly expressed and synergistically promoted EMT transformation.2.14-3-3ζ is capable of inducing changes in the glycosylation levels of OPN through the mediation of CCA cell EMT.Specifically,there is a decrease in the expression of the specific glycan chain GalNAcα(1,3)[αFuc(1,2)]Gal,which is catalyzed by the specific glycosyltransferase GalNAc-T.3.The glycosyltransferase GalNAc-T3 plays an important role in the occurrence and development of CCA and is of great significance for exploring early diagnostic markers and targeted therapies for CCA.4.After 14-3-3ζ-mediated EMT in CCA,OPN O-glycosylation may be regulated by the key molecule GalNAc-T3. |
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